Human immunodeficiency virus (HIV-1), the aetiological agent of AIDS, is distinguished by a genomic structure which is more complex than observed with other animal retroviruses. The genome of HIV-1 has been found to encode the major structural proteins, derived from the gag, pol and env genes, and at least seven non-essential auxiliary proteins derived from the tat, rev, nef, vif, vpu, vpr and tev genes (1,2). Several of these proteins have clearly defined functions; for example, it is known that Tat and Rev regulate viral gene expression, while Vif and Vpu are thought to be required for virion morphogenesis and maturation (3,4,5). However, the function of the other auxiliary proteins such as Nef is more ambiguous.
The nef gene product, a 25-30 kDa protein, is modified by N-terminal myristoylation and phosphorylation and is expressed early in infection (6,7). It is associated with cytoplasmic membrane structures in HIV-infected cells. The Nef protein is encoded by a single open reading frame of the virus genome overlapping with the 3' long terminal repeat sequence (LTR; 8,9). While recent studies of Rhesus monkeys infected with simian immunodeficiency virus have suggested an important role for Nef in the maintenance of high virus load and in viral pathogenesis (10), and while prolonged survival in HIV-infected patients is correlated with the absence of Nef, the biological function and mechanism of action of Nef remain controversial. Nef has been reported to be a negative regulator of HIV-1 replication (2,10,11): T cells stably transformed for Nef expression demonstrate a delay in virus multiplication (11). Further reports have suggested that Nef may act to repress transcription from the HIV-1 LTR (12,13,14), such that Nef may act to regulate the positive effects of the viral transactivator, Tat. However, other studies have been unable to confirm the inhibitory effects of Nef on viral transcription or replication in vitro, and indeed in one instance showed that Nef enhances viral replication (15).
We have found that Nef possesses no G-protein activities, using highly purified recombinant Nef protein produced in either E. coli or yeast.
The effect of HIV-1 Nef on host-cell function is equally controversial. The major targets for HIV-1 infection are CD4.sup.+ T-lymphocytes (16,17). This cell population, most of which constitutes the T-helper cell category, is intimately involved in the host cell response to infection. Specific interactions of the CD4 antigen with invariant determinants of MHC class II molecules on antigen-presenting cells, in conjunction with recognition of peptide antigen by the T-cell receptor (TCR), initiate the events which lead to the expression of the activated T-cell phenotype. Moreover, it has been proposed that CD4 and its associated tyrosine kinase may play an active role in the CD3/TCR activation process (18,19). By use of a vaccinia virus recombinant expressing the nef gene, or the nef gene under control of a murine retrovirus LTR, it has been found that the protein is capable of down-regulating CD4 cell surface expression in CEM T4 cells (20,21). Down-regulation occurred after translation of CD4 mRNA and before expression of mature protein on the cell surface. Furthermore, there is evidence that virally-encoded Nef protein interferes with a signal emanating from the TCR complex that induces IL-2 gene transcription (22). Hence, rather than affecting viral replication, Nef may act to alter the normal host cell response to infection by impairing signal transduction pathways. However, interpretation of these results requires caution because of findings that cells transduced with the same vector, but with no functional nef gene, also exhibited reduced expression of surface CD4 (23), while the effect of Nef upon IL-2 gene transcription remains to be substantiated.
The mechanisms of activation of T cells and of their stimulation with IL-2 are not yet completely understood. However, it is known that stimulation of activated T cells with IL-2 leads to a rapid, transient increase in the catalytic activity of the enzyme p56.sup.lck, a member of the protein tyrosine kinase (PTK) group of enzymes, accompanied by the phosphorylation of this PTK on multiple serine residues (43). A growing body of evidence suggests that tyrosine and serine/threonine protein kinases, and in particular p56.sup.lck, which are known to be associated with the .beta. chains of IL-2 receptor and of CD4, may be involved in IL-2-dependent proliferative signals (18,44).
In order conclusively to determine possible functions of the HIV-1 Nef protein, we have obtained Nef protein in a highly purified form, and have studied its effects on cellular function. Nef 27 and Nef 25, translated from the first and second initiation codons of the nef gene of HIV-1 clone pNL4.3 respectively, were overproduced in E. coli and purified to homogeneity.
We have also synthesised peptides corresponding to amino acids 2-19 and 2-22 of the N-terminal sequence of Nef. Using these purified proteins and peptides, we have demonstrated that Nef has membrane-perturbing properties, in that it is capable of fusing small unilamellar phospholipid vesicles and of inducing non-lamellar phases in lipid bilayers. These properties appear to reside in the 21 residue N-terminal sequence of Nef, which has considerable homologies across all strains of HIV with the sequence of the membrane-perturbing honey bee venom peptide, melittin.
We have used an electric field electroporation technique termed Baekonization to introduce the highly purified Nef 27 or Nef 25 into various CD4.sup.+ T-cell lines and phytohaemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMC), in order to investigate the effect of these proteins on CD4 cell surface expression and expression of IL-2 receptor (IL-2 R). We have found that the T-cell lines MT-2, CEM and Jurkat, and PHA-activated PBMC, containing Nef 27 after electroporation showed significantly reduced expression of surface CD4. Additionally, MT-2 cells and PBMC containing Nef 27 also demonstrate severely reduced expression of IL-2 R, constitutively in the case of MT-2 cells or after stimulation with PHA in PBMC. In direct contrast, the T-cell lines and PBMC which contain Nef 25 after electroporation expressed unaltered levels of surface CD4 and IL-2 R.
As well as identifying the active region of the Nef protein, we have found that Nef 27 inhibits the proliferative response of PHA-activated PBMC in response to recombinant IL-2, and that Nef 27 is able to inhibit the phosphorylation of p56.sup.lck in PBMC which is induced by IL-2 stimulation, while Nef 25 is not. We have been able to demonstrate an interaction between Nef and p56.sup.lck, which may be involved in the inhibition of cell proliferation and IL-2-dependent phosphorylation of p56.sup.lck by Nef 27.